Gene Cloning and DNA Analysis: An introduction

Transcription

1 Gene Cloning and DNA Analysis: An introduction T. A. Brown. 6th edition 2010 Published by Blackwell Science Ltd & /yclclass/ =>2011 Part I The Basic Principles of Gene Cloning and DNA Analysis 1 Why gene cloning and DNA analysis are important 2 Vectors for gene cloning: plasmids and bacteriophages 3 Purification of DNA from living cells 4 Manipulation of purified DNA 5 Introduction of DNA into living cells 6 Cloning vectors for E. coli 7 Cloning vectors for eukaryotes 8 How to obtain a clone of a specific gene 10 The polymerase chain reaction Part II The Applications of Gene Cloning and DNA Analysis in Research 11 Sequencing genes and genomes 12 Studying gene expression and function 13 Studying genomes Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 14 Production of protein from cloned genes 15 Gene cloning and DNA analysis in medicine 16 Gene cloning and DNA analysis in agriculture 17 Gene cloning and DNA analysis in forensic medicine 1

2 1 Why Gene Cloning and DNA Analysis are Important 1.1 The early development of genetics 1.2 The advent of gene cloning and the polymerase chain reaction 1.3 What is gene cloning? 1.4 What is PCR? 1.5 Why gene cloning and PCR are so important Obtaining a pure sample of a gene by cloning PCR can also be used to purify a gene 1.6 How to find your way through this book Middle of the 19th century: Gregor Mendel A set of rules to explain inheritance of biological characteristics Control by a factor (gene) 1900: rediscovery of Mendel s laws make the birth of genetics 2

3 The early development of genetics 1903: W. Sutton genes reside on chromosomes 1910: T. H. Morgan developed techniques for gene mapping By 1922, analysis of relative positions of >2000 genes on 4 chromosomes of Drosophila melanogaster DNA is the genetic material 1944: Avery, Macleod, & McCarty 1952: Hershey & Chase The early development of genetics Discovery of role of DNA A tremendous stimulus to genetic research Many famous biologists contributed to the second great age of genetics Delbrück, Chargaff, Crick, & Monod structure of DNA was elucidated genetic code cracked processes of transcription & translation described 3

4 The advent of gene cloning and the polymerase chain reaction , a whole new methodology was developed recombinant DNA technology or genetic engineering at their core process of gene cloning sparked another great age of genetics 1990s: led to rapid & efficient DNA sequencing techniques enables structure of individual genes to be determined A culmination ( ) with massive genome sequencing projects (2000) Human project was completed in 2000 The advent of gene cloning and the polymerase chain reaction Procedures for studying regulation of individual genes To understand how aberrations in gene activity can result in human diseases such as cancer Modern biotechnology Put genes to work in production of proteins & other compounds needed in medicine & industrial processes 1980s Excitement engendered by gene cloning revolution was at its height 4

5 The advent of gene cloning and the polymerase chain reaction 1985: Kary Mullis The polymerase chain reaction (PCR) during a drive along coast of California one evening His brainwave was an exquisitely simple technique that acts as a perfect complement to gene cloning PCR has made easier many of the technique that were possible but difficult Dancing to carry naked out when in the gene cloning was used on its ownmind field The advent of gene cloning and the polymerase chain reaction DNA analysis traditional range Medicine, agriculture & biotechnology PCR has extended its range & enabled MB to find new applications New disciplines ( ) Archaeogenetics ( ), molecular ecology ( ), DNA forensics Human evolution, impact of environmental change on biosphere, fight against crime 40 yrs passed, we are still riding rollercoaster &there is no end to excitement in sight. 5

6 What is gene cloning? 1.A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule 2.The vector transports the gene into a host cell which is usually a bacterium, although other types of living cell can be used 3.Within the host cell the vector multiplies producing numerous identical copies not only of itself but also of the gene that it carries What is gene cloning? Transformation 42 C, 90 6

7 What is gene cloning? 4. When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny ( ) & further vector replication takes place 5. After a large number of cell divisions, a colony, or clone, of identical host cells is produced Each cell in the clone contains one or more copies of the recombinant DNA moleculeprogeny ( ) The gene carried by the recombinant molecule is now said to be cloned What is gene cloning? 7

8 What is PCR? 1.The mixture is heated to 94 C The hydrogen bonds are broken, causing the molecules to denature 2.The mixture is cooled down to C Two strand of each molecule could join back together, but most do not Because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions What is PCR? 8

9 What is PCR? 3. The temperature is raised to 74 C Mixture: Taq DNA polymerase, dntp It attaches to one end of each primer & synthesizes new strands of DNA, complementary to template DNA 2 => 4 strands 4. Temperature is increased back to 94 C Denature again A second cycle of denaturation-annealing-synthesis (4 => 8 strands) Repeat 30 cycles: >130 million (10 6 ) new ds molecules Why gene cloning and PCR are so important? Both techniques can provide a pure sample of an individual gene, separated from all the other gene in the cell 9

10 Obtaining a pure sample of a gene by cloning DNA fragment to be cloned is one member of a mixture of many different fragments This mixture could indeed be the entire genetic complement of an organism Each fragments becomes inserted into a different vector to produce a family of recombinant DNA molecule One of which carries the gene of interest Only one recombinant DNA molecules is transported into any single host cell The gene is now separated away from all the other gene in the original mixture Figure 1.3 Cloning allows individual A fragments of DNA to be purified B C cloning ABC amplify A B C A B C ABC 10

11 Figure 1.4 The problem of selection >4000 different genes in genome of E. coli 5X as many genes in human genome How to separated from all the other gene Success or failure of a gene cloning Ability to identify the particular clone of interest from the many different ones that are obtained Selection Chap 8: a variety of different strategies can be used Once a gene has been cloned there is almost no limit to the information that can be obtained about it structure & expression Methods for studying the structure & expression of a cloned gene are described in chap 10 & 11 11

12 PCR can also be used to purify a gene Figure 1.5 Gene isolation by PCR Outcome is the same as with a gene cloning Automatically selected A rapid technique Complete in a few hours Cloning: months Much less complicated Tremendous sensitivity PCR has two limitations The sequences of these annealing sites must be known PCR cannot be used to isolate genes that have not been studied before That has to be done by cloning The length of DNA sequence that can be copied by PCR 5 kb: copy fairly easily < 40 kb: using specialized techniques > 40 kb: Many genes of human & other vertebrates Cloning must be used Gene cloning is the only way of isolating long genes or those that have never been studied before. 12

13 Clinical applications of PCR Even if the sequence of a gene is not known A gene that has been isolated & sequenced from mouse could therefore be used to design a pair of primers for isolation of the equivalent gene from humans PCR => sequenced A PCR of human globin genes is used to test for the presence of mutations Thalassaemia, sickle cell anemia Detect viruses at earliest stages of an infection Use of primers specific for the DNA of a diseasecausing virus Even if there is just one DNA molecule in starting mixture How to find you way through this book Part I: we deal with the basic principle Chap2-9 Techniques for handling DNA Part II: how genes and genome are studied Part III: broader applications Biotechnology, medicine, agriculture & forensic science 13

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